To design rational inhibitors of essential HIV regulatory proteins, it is important to understand their mechanism of action, including specific interactions with key host cell proteins. We recently identified a human RNA-binding protein, TRP-185 (TRP-1), which is important for transcriptional regulation of the HIV-1 promoter by the viral regulatory protein, Tat. Preliminary data based on transcriptional activation by Tat in vitro suggest that TRP-185 functions primarily to recruit Tat to the promoter via specific binding to the loop of the TAR RNA element, and that this mechanism of attracting Tat to the HIV promoter is of greater fundamental importance than is direct binding of Tat to TAR RNA. In this proposal, we seek to establish additional direct evidence that TRP- 185 is critical for Tat function through TAR by examining the effect of adding highly purified fractions of TRP-185 to reconstituted transcription systems that have been depleted of TRP-185, or by adding TRP-185 to Drosophila embryo transcription extracts, which lack endogenous TRP-185. Control experiments will be carried out in parallel using a GAL4/Tat fusion protein and a synthetic HIV-1 promoter that contains multiple GAL4 binding sites and lacks the TAR element, which behaves as a model for TAR-independent trans-activation by Tat and therefore should be unresponsive to the TRP-185. We propose to isolate and sequence proteolytic fragments of purified TRP-185, and to use this information to clone the gene. Sequence analysis of the gene will reveal the identity and nature of TRP-185, and antisera derived from expressed fragments of the cloned gene will be used to inhibit the TRP-185/TAR RNA complexes, thereby confirming the identity o the gene. Additional experiments are proposed to further purify TRP-CoF, a protein which is required for stable binding of TRP-185 to TAR, and to determine whether TRP-CoF and TRP-185 interact directly with each other in the absence of TAR RNA. Finally, we will examine whether TRP-185 (or other cellular proteins) interact specifically with Tat by means of affinity chromatography. Taken together, these data should greatly strengthen our understanding of the role of TRP-185 and other cellular factors in TAR- dependent trans-activation by Tat.